U.S. patent number 11,279,079 [Application Number 17/034,261] was granted by the patent office on 2022-03-22 for cell electrochemical sensor based on 3d printing technology and application thereof.
This patent grant is currently assigned to Jiangnan University. The grantee listed for this patent is Jiangnan University. Invention is credited to Jian Ji, Jiadi Sun, Xiulan Sun, Kaimin Wei, Yinzhi Zhang.
United States Patent |
11,279,079 |
Sun , et al. |
March 22, 2022 |
Cell electrochemical sensor based on 3D printing technology and
application thereof
Abstract
The disclosure relates to a cell electrochemical sensor based on
a 3D printing technology and application thereof and belongs to the
technical field of electrochemical sensors and toxin detection. The
cell electrochemical sensor of the disclosure is constructed based
on a 3D printing technology, and the construction method comprises
the following steps: precisely depositing a cell/carbon
nanofiber/GelMA composite hydrogel on a working electrode of a
screen-printed carbon electrode through 3D printing, and carrying
out curing to obtain the cell electrochemical sensor. The
disclosure constructs a cell electrochemical sensor with a
three-dimensional cell growth environment and rapid and sensitive
response. The cell electrochemical sensor constructed by the
disclosure can be used for quickly and effectively determining the
combined effect type and effect degree of deoxynivalenol family
toxins by combining an electrochemical impedance method and a
combination index method.
Inventors: |
Sun; Xiulan (Wuxi,
CN), Sun; Jiadi (Wuxi, CN), Ji; Jian
(Wuxi, CN), Zhang; Yinzhi (Wuxi, CN), Wei;
Kaimin (Wuxi, CN) |
Applicant: |
Name |
City |
State |
Country |
Type |
Jiangnan University |
Wuxi |
N/A |
CN |
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Assignee: |
Jiangnan University (Wuxi,
CN)
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Family
ID: |
71812569 |
Appl.
No.: |
17/034,261 |
Filed: |
September 28, 2020 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20210023770 A1 |
Jan 28, 2021 |
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Foreign Application Priority Data
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Apr 1, 2020 [CN] |
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202010249330.X |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N
33/5014 (20130101); B33Y 80/00 (20141201); G01N
27/3278 (20130101); G01N 27/308 (20130101); B33Y
10/00 (20141201); B29C 64/106 (20170801); B29K
2307/04 (20130101); B29K 2089/00 (20130101); B29K
2105/124 (20130101) |
Current International
Class: |
G01N
27/327 (20060101); G01N 33/50 (20060101); B33Y
80/00 (20150101); B33Y 10/00 (20150101); B29C
64/106 (20170101) |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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102944598 |
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Feb 2013 |
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CN |
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203178279 |
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Sep 2013 |
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CN |
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106645344 |
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May 2017 |
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CN |
|
107219274 |
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Sep 2017 |
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CN |
|
Other References
Donglei Jiang et al., "A novel electrochemical mast cell-based
paper biosensor for the rapid detection of mil allergen casein",
Biosensors and Bioelectronics 130 (2019)299-306, Jan. 30, 2019
(Year: 2019). cited by examiner .
Thomas Robert Heinrich Buch, et al., "Functional expression of the
transient receptor potential channel TRPA1, a sensor for toxic lung
inhalants, in pulmonary epithelial cells", Chemico-Biological
Interactions, vol. 206, Issue 3, Dec. 5, 2013, pp. 462-471 (Year:
2013). cited by examiner .
Ddonglei Jiang et. al., "A novel electrochemical mast cell-based
paper biosensor for the rapid detection of mil allergen casein",
Biosensors and Bioelectronics 130 (2019)299-306, Jan. 30, 2019.
cited by applicant .
Su Ryon Shin, et. al., "Carbon nanotube reinforced hybrid microgels
as scaffold martials for cell encapsulation", ACS Nano, vol. 6 No.
1, 362-372, Nov. 26, 2011. cited by applicant.
|
Primary Examiner: Huson; Monica A
Assistant Examiner: Grace; Kelsey C
Attorney, Agent or Firm: IPro, PLLC
Claims
What is claimed is:
1. A method for making a cell electrochemical sensor, wherein the
method is based on a 3D printing technology comprising the
following steps: preparing a cell/carbon nanofiber/gelatin
methacryloyl (GelMA) composite hydrogel by mixing a carbon
nanofiber solution with a GelMA solution to obtain a carbon
nanofiber/GelMA composite solution, and then uniformly mixing cells
with the carbon nanofiber/GelMA composite solution to obtain the
cell/carbon nanofiber/GelMA composite hydrogel; and depositing the
cell/carbon nanofiber/GelMA composite hydrogel obtained on a
working electrode of a screen-printed carbon electrode by 3D
printing, and followed by curing to obtain the cell electrochemical
sensor; wherein a final concentration of GelMA in the cell/carbon
nanofiber/GelMA composite hydrogel is 5% to 15%; wherein a final
concentration of cells in the cell/carbon nanofiber/GelMA composite
hydrogel is 1.times.10.sup.6/mL to 1.times.10.sup.7/mL; and wherein
the cells are lung adenocarcinoma epithelial cells A549.
2. The method of claim 1, wherein the screen-printed carbon
electrode is coated with gold nanoparticles, and wherein the method
further comprises irradiating the coated screen-printed carbon
electrode and carbon nanofibers with ultraviolet light prior to
depositing the cell/carbon nanofiber/GelMA composite hydrogel
obtained on the working electrode of the screen-printed carbon
electrode.
3. The method of claim 1, wherein the screen-printed carbon
electrode is printed on PET as a substrate.
4. The method of claim 1, wherein the screen-printed carbon
electrode is a circle with the diameter of 3 mm and a printing
layer height of 0.3 mm.
5. The method of claim 1, wherein curing comprises irradiation of
the cell electrochemical sensor with light source comprising a
wavelength of 405 nm.
6. The method of claim 1, wherein preparing the cell/carbon
nanofiber/gelatin methacryloyl (GelMA) composite hydrogel further
comprises adding lithium phenyl-2,4,6-trimethylbenzoylphosphinate
(LAP), GelMA, and carbon nanofiber to a cell culture medium to form
the carbon nanofiber/GelMA composite solution, and then adding the
cells to the carbon fiber/GelMA composite solution to obtain the
cell/carbon nanofiber/GelMA composite hydrogel.
Description
TECHNICAL FIELD
The disclosure relates to a cell electrochemical sensor based on a
3D printing technology and application thereof and belongs to the
technical field of electrochemical sensors and toxin detection.
BACKGROUND
3D printing technology is a computer-aided technology which
produces engineering tissue in a mechanized, organized and
optimized way, can assemble tissue by precisely positioning
biological materials and living cells layer by layer, and has
spatial control capabilities. 3D printing can be used not only to
make basic arrays, but also to develop more complex arrays by
setting different stay times or repeating specific G codes without
making special molds or masks, which enables bioprinting of
three-dimensional tumor array chips meeting a series of specific
drug screening requirements for rapid on-demand drug screening.
For a long time, the pollution problem of mycotoxins in food and
feed has always been serious, the global food and feed loss caused
by fungal pollution accounts for 20%-30% of the total output, the
deoxynivalenol-family mycotoxins are the mycotoxins with the
highest detection rate in the northern hemisphere, the most serious
one is deoxynivalenol (DON), and the main polluted crops comprise
wheat, corn and the like. DON is heat-resistant, acid-resistant and
storage-resistant. The structure of DON cannot be destroyed by
general heat treatment processing methods, and after humans and
animals eat food containing DON, different degrees of toxic
reactions, such as vomiting and loss of appetite can be caused. In
addition to DON, acetylated derivatives (such as 3-ADON, 15-ADON)
of DON are often detected in grains such as wheat and corn. Both
3-ADON and 15-ADON belong to the deoxynivalenol-family mycotoxins.
It is shown through in-vivo experiments in pigs that 3-ADON can be
quickly converted into DON in blood; data shows that acetylated DON
can be absorbed by intestinal tissue faster and is considered to be
more toxic. Based on the high toxicity and high detection rate of
DON, countries and regions have established clear limit standards
for DON. The EU defines the limit standard of DON in different
foods to be 200-1750 .mu.g/kg. China defines in the national
standard G2762-2017 that DON in grains and products thereof shall
not exceed 1000 .mu.g/kg. Current researches on the toxicity of
toxins often focus on a single toxin, and there is a lack of
comprehensive studies on the types and mechanisms of combined
effects of mycotoxins.
Current toxicity evaluation methods mainly rely on two-dimensional
cell experiments and animal experiments. Two-dimensional cell
experiment methods have a low cost, a short cycle and certain
homology with the body, but two-dimensional cell culture
environments have greater differences from human body environments.
Although results of animal toxicology experiments can truly,
comprehensively and systematically reflect the effects of drugs on
the body, there are disadvantages of high cost, long cycle,
unsatisfactory repeatability, etc.
With the development and progress of science and technology, a
variety of new technologies and methods combining traditional cell
and sensor technologies provide more new methods for the study of
toxicity mechanisms. In the construction of cell sensors, cells
serving as receptors are immobilized to the interface. When the
cells are stimulated by external drugs, changes in cell
physiological activity can be caused. These changes can be
converted into photoelectric signals. The magnitude of the signal
changes can be used in qualitative and quantitative analysis of
drug stimulation of cells. Currently, the patent (CN 107219274 B)
discloses a preparation method of a cell electrochemical sensor for
analyzing the combined toxicity of mycotoxins. The method
specifically comprises the steps of immobilizing laminin to the
surface of an electrode, then inoculating the electrode surface
with cells, and dropwise adding rat tail collagen to form a 3D
complex to immobilize the cells to obtain the cell sensor. The
sensor prepared in this way may have problems of uneven
distribution and low detection precision.
SUMMARY
In order to solve at least one of the problems above, the
disclosure provides a construction method of a cell electrochemical
sensor based on a 3D printing technology and application of the
sensor in combined toxicity evaluation of deoxynivalenol-family
mycotoxins. The disclosure uses the combination of 3D printing and
an electrochemical cell sensor to construct a three-dimensional
lung adenocarcinoma epithelial cell culture system which is
reliable, easy to operate and high in reproducibility. The
impedance value is measured by an electrochemical AC impedance
method to judge the damage conditions of cells after the cells are
stimulated by toxins so as to quickly and effectively evaluate the
cytotoxicity of mycotoxins. The combination index method (CI) is
combined to analyze the combined toxicity of two or more toxins and
determine the combined effect type.
The first objective of the disclosure is to provide a method for
preparing a cell electrochemical sensor. The cell electrochemical
sensor is constructed based on a 3D printing technology. The
construction method comprises the following steps:
(1) preparing a cell/carbon nanofiber/GelMA composite hydrogel: a
carbon nanofiber solution is added into a gelatin methacryloyl
(GelMA) solution for uniform mixing to obtain a carbon
nanofiber/GelMA composite solution; then cells are uniformly mixed
with the carbon nanofiber/GelMA composite solution to obtain the
cell/carbon nanofiber/GeMA composite hydrogel; and
(2) precisely depositing the cell/carbon nanofiber/GelMA composite
hydrogel on a working electrode of a screen-printed carbon
electrode through 3D printing, and carrying out curing to obtain
the cell electrochemical sensor.
In an embodiment of the disclosure, the final concentration of
GelMA in the cell/carbon nanofiber/GelMA composite hydrogel is
5-15%, preferably 5-7.5%.
In an embodiment of the disclosure, the final concentration of
cells in the cell/carbon nanofiber/GelMA composite hydrogel is
1.times.10.sup.6-1.times.10.sup.7/mL.
In an embodiment of the disclosure, the final concentration of
carbon nanofibers in the cell/carbon nanofiber/GelMA composite
hydrogel is 0.5-1 mg/mL.
In an embodiment of the disclosure, the carbon nanofibers are
purchased from Xianfeng Nano, the model is XFM60, the diameter is
200-600 nm, and the length is 5-50 m.
In an embodiment of the disclosure, the cells are lung
adenocarcinoma epithelial cells A549.
In an embodiment of the disclosure, 3D printing specifically
comprises: pouring the cell/carbon nanofiber/GelMA composite
hydrogel into a printing syringe, setting an initial needle
position, a syringe temperature, a working platform temperature, an
extrusion pressure, a graphic size, a graphic layer number and a
nozzle walking speed, then precisely depositing the composite
hydrogel on the working electrode of the screen-printed carbon
electrode through 3D printing, and curing the composite hydrogel
under a portable curing light source.
In an embodiment of the disclosure, a three-dimensional structure
model of 3D printing is a circle with the diameter of 3 mm, the
printing layer number is 1, and the printing layer height is 0.3
mm; the printing cylinder temperature is 23-27.degree. C., the
platform temperature is 1-5.degree. C., the needle inner diameter
is 0.26 mm, the extrusion pressure is 0.08-0.2 Mpa, and the nozzle
walking speed is 180-300 mm/min.
In an embodiment of the disclosure, a bioprinted cell and hydrogel
complex is irradiated with a portable curing light source with the
wavelength of 405 nm for 10-20 seconds for curing.
In an embodiment of the disclosure, a preparation method of the
cell/carbon nanofiber/GelMA composite hydrogel specifically
comprises:
(1) preparation of a gelatin methacryloyl (GelMA) solution: an LAP
initiator is added into a DMEM cell culture medium for well mixing
and dissolving, and then a GeMA material is added into the
dissolved standard initiator solution for dissolving in a water
bath in the dark to obtain the GelMA solution; and
(2) preparation of the cell/carbon nanofiber/GelMA composite
hydrogel: a carbon nanofiber solution is added into the gelatin
methacryloyl (GelMA) solution for uniform mixing to obtain a carbon
nanofiber/GelMA composite solution; then cells are uniformly mixed
with the carbon nanofiber/GelMA composite solution to obtain the
cell/carbon nanofiber/GeMA composite hydrogel.
In an embodiment of the disclosure, the LAP standard initiator
solution in step (1) is prepared with a DMEM cell culture medium,
the mass concentration of the LAP standard initiator solution is
0.5%, the dissolution temperature is 50-60.degree. C., and the
dissolution time is 15-30 minutes; the mass concentration of the
GeMA solution is 5%-15%, the dissolution temperature is
50-60.degree. C., and the dissolution time is 15-30 minutes; after
being completely dissolved, the GelMA solution is filtered by a
0.22 .mu.m sterile filter membrane for sterilization.
In an embodiment of the disclosure, a preparation method of the
carbon nanofiber solution in step (2) comprises: adding a certain
amount of carbon nanofibers into a phosphate buffer solution (PBS)
to make the concentration of carbon nanofibers reach 1-2 mg/mL, and
carrying out ultrasonic treatment for 2 hours to obtain the carbon
nanofiber solution.
In an embodiment of the disclosure, in step (2), the final
concentration of cells is adjusted to be
1.times.10.sup.6-1.times.10.sup.7/mL, and the final concentration
of carbon nanofibers is 0.5-1 mg/mL.
In an embodiment of the disclosure, after the carbon nanofiber
solution is prepared and the screen-printed carbon electrode is
electroplated with gold nanoparticles, both the carbon nanofiber
solution and the screen-printed carbon electrode need to be placed
under an ultraviolet light for irradiation for 2 hours or above for
sterilization treatment.
In an embodiment of the disclosure, the screen-printed carbon
electrode is a modified screen-printed carbon electrode obtained by
modifying and electroplating the screen-printed carbon electrode
with gold nanoparticles.
In an embodiment of the disclosure, the gold nanoparticles are
prepared from 1 mL of 1% chloroauric acid, 1 mL of 1 mmol/L
sulfuric acid solution and 8 mL of ultrapure water. The gold
nanoparticles are deposited on the screen-printed carbon electrode
by an electroplating method, the electroplating voltage is -0.2-0.4
V, and the electroplating time is 120-160 seconds.
The second objective of the disclosure is a cell electrochemical
sensor prepared by the method of the disclosure, and the
cell/carbon nanofiber/GeMA composite hydrogel is immobilized on the
surface of the working electrode in the cell electrochemical
sensor.
In an embodiment of the disclosure, the final concentration of
GelMA in the cell/carbon nanofiber/GelMA composite hydrogel is
5-15%, preferably 5-7.5%.
In an embodiment of the disclosure, the final concentration of
cells in the cell/carbon nanofiber/GelMA composite hydrogel is
1.times.10.sup.6-1.times.10.sup.7/mL.
In an embodiment of the disclosure, the final concentration of
carbon nanofibers in the cell/carbon nanofiber/GelMA composite
hydrogel is 0.5-1 mg/mL.
The third objective of the disclosure is to provide a method for
evaluating the toxicity of deoxynivalenol-family mycotoxins by
using the cell electrochemical sensor of the disclosure. The method
is as follows: one toxin is used to stimulate the cell sensor alone
or two or more toxins are combined to act on the cell sensor, and
then the electrochemical AC impedance method and the combination
index method are used to analyze the cytotoxicity or the combined
effect type.
In an embodiment of the disclosure, the cell sensor needs to be
incubated before application, and specific operations comprise:
dropping a DMEM cell culture medium at a working electrode of a
screen-printed carbon electrode to ensure that the culture medium
can cover a cell/carbon nanofiber/GelMA complex on the working
electrode to provide cells with nutrients needed for growth, and
then placing the working electrode in an incubator with the carbon
dioxide concentration of 5% and the humidity of 95% for incubation
at 37.degree. C. for 6-12 hours; after incubation, removing the
original culture medium on the working electrode, diluting toxin
standard substances with the DMEM cell culture medium into gradient
concentration solutions, dropping the gradient concentration
solutions on the working electrode printed with cells, and then
carrying out electrochemical detection after placing the working
electrode in the incubator for effect for 24 hours.
In an embodiment of the disclosure, the deoxynivalenol-family
mycotoxins are one or two of deoxynivalenol (DON) and
15-acetyl-deoxynivalenol (15-ADON).
In an embodiment of the disclosure, the method uses a modified
screen-printed carbon electrode as a sensing interface, a PET
material is used as a substrate, the working electrode is composed
of carbon, the reference electrode is composed of silver/silver
chloride, the counter electrode is composed of carbon,
electrochemical detection adopts electrochemical impedance
spectroscopy (EIS) to test electrical signal changes of cells
stimulated by toxins, the initial potential is 0.2 V, and the
frequency range is 1 Hz-10.sup.5 Hz.
In an embodiment of the disclosure, the electrochemical AC
impedance method (EIS) measures the signals obtained by the
electrochemical sensor stimulated by different concentrations of
toxins, the impedance value is calculated by fitting the equivalent
circuit with Zview software, the cell viability inhibition rates of
different concentrations of toxins are calculated, and the
calculation method is as follows:
.times..times..times..times..times..times..times..times..times..times.
##EQU00001##
wherein R.sub.dosing refers to the impedance value of the
screen-printed carbon electrode (SPE) with toxin stimulation and
bioprinting modification, R.sub.0dosing refers to the impedance
value of the screen-printed carbon electrode (SPE) with bioprinting
modification and without toxin stimulation, and R.sub.carbon
nanofiber/GelMA refers to the impedance value of the SPE with
modification of carbon nanofibers and GelMA hydrogel and without
cells.
In an embodiment of the disclosure, a combined effect formula in
the combination index method is:
##EQU00002##
wherein f.sub.a refers to a cell damage effect rate, f refers to a
cell undamaged effect rate, D refers to a toxin concentration,
D.sub.m refers to a toxin concentration when the cell damage effect
rate reaches 50%, and m refers to a dose-effect curve
coefficient.
In an embodiment of the disclosure, a calculation formula of the CI
index of the combination index method is:
.times. ##EQU00003##
wherein (D).sub.j refers to the required concentration when x %
damage effect is caused by the combined effect of toxins,
(D.sub.x).sub.j refers to the concentration when x % damage effect
is caused by a single toxin, and if CI>0.9, the combined effect
type of toxins is considered to be antagonism; if CI=0.9-1, the
combined effect type of toxins is considered to be an additive
effect; and if CI<0.9, the combined effect type of toxins is
considered to be synergism.
The fourth objective of the disclosure is application of the cell
electrochemical sensor in the fields of drug development,
toxicology testing and environmental monitoring.
Compared with the prior art, the disclosure has the following
advantages:
(1) In the disclosure, carbon nanofibers are added into GeMA to
improve the conductivity of the hydrogel, so that the prepared
sensor has higher sensitivity for the detection of toxins.
(2) In the disclosure, the screen-printed carbon electrode is used
as the sensing interface, the constructed cell sensor chip has the
advantages of small size, portability, low sample requirement
amount, high detection speed, mass production and modification and
the like, and the problem of cross pollution in practical
application is avoided.
(3) The cell sensor of the disclosure can be used for determining
the toxic effect degrees of two or more deoxynivalenol-family
mycotoxins. For a long time, China's food and feed have been
seriously polluted by mycotoxins, and pollution caused by multiple
toxins at the same time also happens. The disclosure can be used
for not only determining the cytotoxicity of a single toxin and two
or more toxins, but also further determining the combined effect
type of the toxins, and references can be provided for the
determination of relevant testing standards.
(4) In the disclosure, a three-dimensional cell culture system is
constructed through reasonable combination of a 3D printing
technology and the cell electrochemical sensing field. The method
is convenient and reliable to operate, human errors are reduced to
a certain extent, a new method and idea are provided for evaluating
the toxicity of mycotoxins, a more realistic and effective
evaluation method is also provided for the study of combination
toxicity mechanisms, and the cell sensor is expected to be applied
in the fields of food safety, biomedicine and the like.
BRIEF DESCRIPTION OF FIGURES
FIG. 1: A schematic diagram of the modification process of a
screen-printed electrode.
FIG. 2: A schematic diagram of a 3D printing process for preparing
a cell electrochemical sensor.
FIG. 3: Electrochemical characterization diagrams of a construction
process of the cell electrochemical sensor, wherein FIG. 3A is an
electrochemical signal obtained by cyclic voltammetry (CV); FIG. 3B
is an electrochemical signal obtained by differential pulse
voltammetry (DPV); FIG. 3C is an electrochemical signal obtained by
electrochemical impedance spectroscopy (EIS); in FIGS. 3A-3C, a
refers to a bare electrode (SPCE), b refers to an electrode
modified with AuNPs (AuNP/SPCE), c refers to a carbon
nanofiber/GeMA/AuNPs electrode (CN/GelMA/AuNP/SPCE), and d refers
to a cell/carbon nanofiber/GeMA/AuNPs electrode
(cells/CN/GelMA/AuNP/SPCE).
FIG. 4 is electrode-modified electron microscope characterization
diagrams, wherein FIG. 4A refers to a bare electrode (SPCE); FIG.
4B refers to an electrode modified with AuNPs (AuNP/SPCE-modified
electrode); FIG. 4C refers to a cell/carbon nanofiber/GeMA/AuNPs
electrode (cells/CN/GelMA/AuNP/SPCE-modified electrode); FIG. 4D
refers to cells growing in clusters in a cell/carbon nanofiber/GeMA
composite hydrogel.
FIG. 5: EIS detection result diagrams of the cell sensor when the
cell electrochemical sensor is used for evaluating DON, 15-ADON and
the combined effect of the two toxins, wherein FIG. 5A is an EIS
detection result diagram of the cell sensor after 24 hours of DON
stimulation, and a-f refer to the toxin concentrations of 0.1, 0.2,
0.5, 1, 2 and 5 .mu.g/mL respectively; FIG. 5B is an EIS detection
result diagram of the cell sensor after 24 hours of 15-ADON
stimulation, and a-f refer to the toxin concentrations of 0.1, 0.2,
0.5, 1, 2 and 5 .mu.g/mL respectively; FIG. 5C is an EIS detection
result diagram of the cell sensor after 24 hours of DON and 15-ADON
stimulation, and a-f refer to the toxin concentrations of 0.1+0.1,
0.2+0.2, 0.5+0.5, 1+1, 2+2 and 5+5 .mu.g/mL respectively; FIG. 5D
is a correlation curve of different concentrations of DON and the
electrochemical impedance value of the constructed sensor; FIG. 5E
is a correlation curve of different concentrations of 15-ADON and
the electrochemical impedance value of the constructed sensor; and
FIG. 5F is a correlation curve of different concentrations of
DON+15-ADON and the electrochemical impedance value of the
constructed sensor.
FIG. 6: Experimental comparison results of the cell electrochemical
sensor and a CCK8 method in evaluating the cytotoxicity, wherein
FIG. 6A is DON; FIG. 6B is 15-ADON; and FIG. 6C is the combined
effect of the two toxins DON and 15-ADON.
FIG. 7: An impedance spectrum diagram of electrochemical sensors
prepared with different concentrations of the lung adenocarcinoma
epithelial cells A549, wherein the cell concentration ranges of a-e
are 1.times.10.sup.3/mL, 1.times.10.sup.4/mL, 1.times.10.sup.5/mL,
1.times.10.sup.6/mL and 1.times.10.sup.7/mL respectively.
FIG. 8: FIG. 8A refers to the survival rate of the lung
adenocarcinoma epithelial cells A549 in different concentrations of
GelMA hydrogels; FIG. 8B is a DPV detection result diagram of the
electrode when different concentrations of GelMA hydrogels are
printed on the screen-printed carbon electrode, wherein a-e refer
to the GeMA concentrations of 5%, 7.5%, 10%, 12.5% and 15%
respectively.
FIG. 9: FIG. 9A is an EIS detection result diagram of the cell
sensor when the DON toxin stimulates the cell electrochemical
sensor prepared by a dropping method, wherein a-f refer to the DON
toxin concentrations of 0.1, 0.5, 2, 0.2, 1 and 5 .mu.g/mL
respectively; and FIG. 9B is a correlation curve graph of the DON
toxin and the sensor impedance value.
DETAILED DESCRIPTION
The preferred examples of the disclosure will be described below.
It should be understood that the examples are used for better
explaining the disclosure and are not intended to limit the
disclosure. The carbon nanofibers used in the following examples
and comparative example are purchased from Xianfeng Nano, the model
is XFM60, the diameter is 200-600 nm, and the length is 5-50
.mu.m.
Example 1 Preparation of a Cell Electrochemical Sensor
A method for constructing a cell electrochemical sensor based on a
3D printing technology (shown as FIG. 2) comprises the following
steps:
(1) Preparation of a gelatin methacryloyl (GelMA) solution: An LAP
initiator is added into a DMEM cell culture medium to make the
final concentration reach 0.5%, and then dissolving is carried out
in a water bath at 60.degree. C. for 30 min in the dark to obtain a
dissolved standard initiator solution. A GelMA material is added
into the dissolved standard initiator solution for dissolving in a
water bath at 60.degree. C. for 30 min in the dark, and shaking is
carried out 3 times during the period to obtain a GelMA solution
(the mass concentration is 7.5%); and then the obtained GeMA
solution is filtered with a 0.22 m sterile filter membrane into a
clean container for use.
(2) Preparation of a cell/carbon nanofiber/GelMA composite
hydrogel: A certain amount of carbon nanofibers are added into a
phosphate buffer solution (PBS) to make the concentration of carbon
nanofibers reach 1 mg/mL, ultrasonic treatment is carried out for 2
hours, and then a prepared carbon nanofiber solution is placed
under an ultraviolet light for irradiation overnight. A certain
amount of the carbon nanofiber solution is added into the GeMA
solution obtained by filtration in step (1), the mixed solution is
thoroughly mixed to make the GeMA concentration reach 7.5% and the
carbon nanofiber solid filling amount reach 0.05%, and a carbon
nanofiber/GelMA composite solution is obtained. The human lung
adenocarcinoma epithelial cells A549 are uniformly mixed with the
carbon nanofiber/GelMA composite solution, and the cell
concentration is adjusted to be 1.times.10.sup.6/mL to obtain the
cell/carbon nanofiber/GeMA composite hydrogel.
(3) Modification and electroplating of a screen-printed carbon
electrode with gold nanoparticles (shown as FIG. 1): The
screen-printed carbon electrode needs to be activated in a 0.5
mmol/L sulfuric acid solution first and then scanned by cyclic
voltammetry, the voltage is -0.2 V-0.6 V, the scanning rate is 100
mV/s, and the scanning circle number is 8. The activated
screen-printed carbon electrode is immersed in a 10 mL of a gold
electroplating solution containing 1% chloroauric acid and 1 mmol/L
sulfuric acid solution, a time-current method is adopted, the
electroplating voltage is -0.3 V, the electroplating time is 120
seconds, and then the electrode is rinsed with ultrapure water,
dried with nitrogen and placed under an ultraviolet light overnight
for irradiation and sterilization to obtain the screen-printed
carbon electrode modified and electroplated with gold
nanoparticles.
(4) 3D printing: The cell/carbon nanofiber/GelMA composite hydrogel
prepared in step (2) is poured into a printing syringe, the syringe
temperature is set to be 26.degree. C., the working platform
temperature is 3.degree. C., the extrusion pressure is 0.1 MPa, the
graphic length is 3 mm, the graphic width is 3 mm, the printing
layer number is 1, the nozzle walking speed is 300 mm/min, the
screen-printed carbon electrode modified and electroplated with
gold nanoparticles prepared in step (3) is placed at a specific
position on the working platform, a printing procedure is run to
precisely deposit the composite hydrogel on the working electrode
of the screen-printed carbon electrode, and the composite hydrogel
is irradiated with a portable curing light source with the
wavelength of 405 nm for 10-20 seconds for curing to obtain the
cell electrochemical sensor.
The cell electrochemical sensor obtained in step (4) needs to
undergo cell incubation before application, and specific steps
comprise: dropping a 150 .mu.L of the DMEM cell culture medium at
the working electrode of the screen-printed carbon electrode to
ensure that the culture medium can cover a cell/carbon
nanofiber/GelMA complex on the working electrode to provide cells
with nutrients needed for growth, and then placing the working
electrode in an incubator with the carbon dioxide concentration of
5% and the humidity of 95% for incubation at 37.degree. C. for 6
hours to obtain the incubated cell electrochemical sensor.
Cyclic voltammetry, differential pulse voltammetry, an AC impedance
method and scanning electron microscope characterization are
performed on the cell electrochemical sensor after incubation. The
conditions of the voltammetry method are: the voltage of -0.2 V-0.6
V and the scanning rate of 100 mV/s; the condition of the AC
impedance method is: the frequency range of 1 Hz-10.sup.5 Hz. In
the scanning electron microscope characterization, samples
containing the cells A549 are tested after the cells are cultured
in the CN/GeMA composite hydrogel for 48 hours.
As shown in FIG. 3, compared with a bare screen-printed carbon
electrode, the electrical signal of the gold-plated screen-printed
carbon electrode is significantly enhanced, and it is indicated
that the gold nanoparticles have excellent conductivity. When the
carbon nanofiber/GelMA hydrogel is deposited on the working
electrode, the electrical signal is reduced to some extent. When
the cell/carbon nanofiber/GelMA hydrogel is deposited on the
working electrode, the electrical signal is further reduced due to
the insulation of cell membranes, it is indicated that the cells
are successfully modified on the electrode surface, and preparation
of the cell electrochemical sensor is completed.
The electrode-modified electron microscope characterization diagram
in FIG. 4 shows that AuNPs are evenly distributed on the electrode
surface (FIG. 4B). FIG. 4C shows uniform immobilization of carbon
nanofibers in the GeMA hydrogel, which increases catalytically
active sites for achieving better electrochemical performance.
After the cells A549 are introduced into the screen-printed carbon
electrode, the cells are firmly immobilized and evenly distributed
in the CN/GelMA composite hydrogel. In addition, the cells are also
aggregated in the composite hydrogel, which maintain complete cell
morphologies (FIG. 4D), the good physical state of the cells in the
hydrogel is confirmed, and redox probes are prevented from entering
the electrode surface, so that the electron transfer resistance is
increased.
Example 2 Application of a Cell Electrochemical Sensor Based on a
3D Printing Technology
The incubated electrochemical sensor obtained in example 1 is used
for evaluating the cytotoxicity of deoxynivalenol-family
mycotoxins, and specific operations are as follows:
(1) Drug stimulation: An original culture medium on a working
electrode is removed, toxin standard substances are diluted with a
DMEM cell culture medium into gradient concentration solutions, and
then 150 .mu.L of DON toxin solutions in the concentration range of
0.1, 0.2, 0.5, 1, 2 and 5 .mu.g/mL, 150 .mu.L of 15-ADON toxin
solutions in the concentration range of 0.1, 0.2, 0.5, 1, 2 and 5
.mu.g/mL and 150 .mu.L of DON+15-ADON toxin solutions in the
concentration range of 0.1+0.1, 0.2+0.2, 0.5+0.5, 1+1, 2+2 and 5+5
.mu.g/mL are taken and dropped on the working electrode printed
with cells respectively, the working electrode is placed in an
incubator for effect for 24 hours, and corresponding impedance
values are measured.
(2) Detection of electrochemical signal values: A 150 .mu.L of 2.5
mM Fe(CN).sub.6.sup.3-/4- PBS solution is used as an electrode
reaction system, the frequency range of an electrochemical AC
impedance method (EIS) is 1 Hz-10.sup.5 Hz, and the impedance value
is fitted by Zview software and calculated through the best
equivalent circuit. The toxic effects on cells are generated after
different doses of toxins stimulate the cells, EIS is used for
analyzing the toxicity of the toxins on the lung adenocarcinoma
epithelial cells A549, and a calculation method is as follows:
.times..times..times..times..times..times..times..times..times..times.
##EQU00004##
wherein R.sub.dosing refers to the impedance value of the
screen-printed carbon electrode (SPE) with toxin stimulation and
bioprinting modification, R.sub.0dosing refers to the impedance
value of the screen-printed carbon electrode (SPE) with bioprinting
modification and without toxin stimulation, and R.sub.carbon
nanofiber/GelMA refers to the impedance value of the SPE with
modification of carbon nanofibers and GelMA hydrogel and without
cells.
The combined effect type can be determined by substituting the
inhibition rate obtained after the sensor is stimulated by toxins
into a combination index formula, and a combined effect formula in
the combination index method is:
##EQU00005##
wherein f.sub.a refers to a cell damage effect rate, f.sub.u refers
to a cell undamaged effect rate, D refers to a toxin concentration,
D.sub.m refers to a toxin concentration when the cell damage effect
rate reaches 50%, and m refers to a dose-effect curve
coefficient.
A calculation formula of the CI index is:
.times. ##EQU00006##
wherein (D).sub.j refers to the required concentration when x %
damage effect is caused by the combined effect of toxins,
(D.sub.x).sub.j refers to the concentration when x % damage effect
is caused by a single toxin, and if CI>0.9, the combined effect
type of toxins is considered to be antagonism; if CI=0.9-1, the
combined effect type of toxins is considered to be an additive
effect; and if CI<0.9, the combined effect type of toxins is
considered to be synergism.
(3) Result judgment
TABLE-US-00001 TABLE 1 CI index values obtained after analysis of
the combined effect of mycotoxins by the cell electrochemical
sensor Concentration of Inhibition mycotoxins/(.mu.g/mL) rate/100
Combined DON 15-ADON f.sub.a CI effect type 0.1 0.1 0.0877 1.23033
Antagonism 0.2 0.2 0.2768 0.80967 Synergism 0.5 0.5 0.4027 1.28417
Antagonism 1 1 0.5122 1.79919 Antagonism 2 2 0.6426 2.33635
Antagonism 5 5 0.6778 5.14921 Antagonism
As shown in FIG. 5, the impedance value of the cell sensor prepared
in example 1 is gradually reduced with the increasing dose of DON
in the concentration range of 0.1-5 .mu.g/mL and the increasing
dose of 15-ADON in the concentration range of 0.1-5 .mu.g/mL, the
reason is that after different doses of toxins stimulate the cells
A549, the cells can undergo different degrees of apoptosis, lysis,
and morphological changes, and thus changes of impedance
electrochemical signals are caused. The IC.sub.50 value of DON
measured by the EIS method is 0.9281 .mu.g/mL, the IC.sub.50 value
of 15-ADON is 1.2560 .mu.g/mL, the IC.sub.50 value of DON and
15-ADON during combined effect is 2.279 .mu.g/mL, and specific
results are shown in Table 1. It is found by using the cell
electrochemical sensor constructed in example 1 to analyze the
combined effect type of DON and an acetylated derivative 15-ADON
thereof that the two toxins generally show an antagonistic
effect.
Example 3 A Verification Experiment
The CCK8 method is used for detecting the cytotoxicity of DON,
15-ADON and DON+15-ADON alone and in combination. The lung
adenocarcinoma epithelial cells A549 with the density of
5.times.10.sup.4/mL are adhered to the wall of a 96-well plate for
inoculation, a culture medium is removed after culture for 24
hours, and 100 .mu.L of the toxin solution same as that in example
2 is added. After 24 hours of toxin stimulation, the supernatant is
sucked out, a 100 .mu.L of culture medium containing 10% CCK8 is
added into each well for incubation at 37.degree. C. for 2 hours,
then the absorbance value is measured with a microplate reader at
450 nm, the cell activity inhibition rate is calculated, and the
calculation method is as follows:
.times..times..times..times..times..times. ##EQU00007##
wherein OD.sub.dosing refers to the absorbance value after 24 hours
of toxin stimulation, OD.sub.0dosing refers to the absorbance value
after 24 hours of toxin-free stimulation, and OD.sub.blank refers
to the absorbance value of the pure cell culture medium.
It can be seen from FIG. 6 that the measurement structure of the
cell electrochemical sensor constructed in example 1 for evaluating
the cytotoxicity of vomitoxin is in good consistency with the
results measured by a traditional cytotoxicology method, and the
cell electrochemical sensor can be used for effectively determining
the cytotoxicity of toxins.
Example 4 Optimization of Preparation Parameters of the Cell/Carbon
Nanofiber/GeMA Composite Hydrogel
(1) Final Concentration of Cells
According to example 1, a series of gradient cell concentrations of
cell/carbon nanofiber/GelMA hydrogels are prepared to make the
final concentrations of the lung adenocarcinoma epithelial cells
A549 reach 1.times.10.sup.3/mL, 1.times.10.sup.4/mL,
1.times.10.sup.5/mL, 1.times.10.sup.6/mL and 1.times.10.sup.7/mL
respectively, other parameters remain unchanged, then the
cell/carbon nanofiber/GeMA hydrogels are deposited on the
gold-plated screen-printed carbon electrode through 3D printing to
obtain different cell concentrations of electrochemical sensors,
and electrochemical signals are tested by an AC impedance
method.
As shown in FIG. 7, as the cell concentration is increased, the
blocking effect of cell membranes on the current is improved, and
the impedance value of the cell electrochemical sensor is
increased. When the cell concentration reaches
1.times.10.sup.6/mL-1.times.10.sup.7/mL, the impedance value is no
longer increased significantly, it is indicated that the cells on
the electrode surface are saturated at this time, and the formed
cell electrochemical sensor stays in a relatively stable state.
Therefore, the cell concentration range of
1.times.10.sup.6/mL-1.times.10.sup.7/mL is adopted.
(2) Final Concentration of GeMA
According to example 1, the final GeMA concentrations are adjusted
to 5%, 7.5%, 10%, 12.5% and 15% respectively to prepare cell/GelMA
hydrogels, then the cell/GelMA hydrogels are deposited on the
gold-plated screen-printed carbon electrode through 3D printing, a
calcein-AM/PI cell live and dead double staining kit is used for
detecting the cell viability of cells incubated in different
concentrations of GelMA hydrogels for 72 hours, and differential
pulse voltammetry is used for testing the electrochemical
signals.
A cell live and dead staining experiment is carried out according
to instructions of the kit: the cell/GelMA hydrogel is observed
under a laser confocal microscope after staining, and yellow-green
fluorescent live cells and red fluorescent dead cells are observed
at the same time at an excitation wavelength of 490.+-.10 nm. In
addition, the dead cells are observed separately at an excitation
wavelength of 545 nm, and then cell viability statistics is carried
out through a counting function of an instrument.
As shown in FIG. 8A, the cells A549 have a high survival rate in
the GeMA concentration range of 5-7.5%. It can be seen from FIG. 8B
that as the concentration of GeMA is increased, the current value
of the screen-printed carbon electrode is reduced, the impedance
value is increased, and in order to ensure the activity of the
cells in the sensor and the sensitivity of the sensor, the GelMA
concentration range of 5-7.5% is adopted. If the GeMA concentration
is too low, the requirements of 3D printing cannot be met.
Comparative Example 1
The 3D printing method of example 1 is adjusted to a dropping
method, that is to say, a 10 .mu.L of cell/carbon nanofiber/GelMA
hydrogel is taken and dropped on the screen-printed working
electrode through a pipette, other parameters remained unchanged,
and the cell electrochemical sensor is prepared.
The prepared cell electrochemical sensor is used for
electrochemical detection of DON cell toxicity, and results of EIS
detection and impedance value changing with toxin concentrations
are shown as FIG. 9. It can be seen that there is no regular linear
relationship between the dose of the DON toxin and the impedance
value of the cell electrochemical sensor, and there are large
errors between parallel dose groups. It is indicated that the
deposition of the cells, carbon nanofibers and hydrogel on the
screen-printed carbon electrode in a dropping method can cause the
problem of uneven distribution of cells and materials due to human
errors, thereby reducing the detection precision.
Therefore, the 3D printing method can be used for more precisely
positioning biological materials and living cells, human errors are
reduced to a certain extent, mass production is allowed, and the
method has the advantages of ensuring the precision and being
convenient in operation.
* * * * *